Malaria remains the most important parasitic disease in the world, affecting hundreds of millions of people and killing almost 1 million each year. Anti-malarial drugs remain the mainstay of malaria management, but the emergence and spread of resistance to these drugs is of grave concern. It is apparent that for the foreseeable future we need to feed a robust pipeline with compounds that either overcome drug resistance or target novel physiological processes vital for the parasite. Towards this goal, our laboratory has shown the mitochondrion of malaria parasites to be highly diminished and divergent from its mammalian counterpart, and has validated its physiology as a target for anti-malarial drugs such as atovaquone. We have demonstrated selective inhibition of parasite mitochondrial electron transport chain (mtETC) at the cytochrome bc1 complex by at least 3 additional chemical classes of compounds under development as antimalarials. We showed that a critical function of mtETC in Plasmodium falciparum blood stages was to serve as an electron disposal system for the mitochondrially located dihydroorotate dehydrogenase (DHODH), thereby supporting the essential pyrimidine biosynthesis. Among several implications of this finding, it provided further validation of parasite DHODH as a target for anti-malarial drug development, an effort underway by several groups. Our initial investigations of the ATP synthase complex, ubiquinone-requiring mitochondrial dehydrogenase, and enzymes and architecture of tricarboxylic acid (TCA) metabolism all suggest unusual aspects of mitochondrial functions in malaria parasites. Our results also suggest essential nature of some of these processes with the possibility that they could serve as potential targets for drug development. With greatly improved tools and technology developed over the recent years, we propose to investigate these functions in greater details with the hope that this knowledge could guide search for novel strategies to develop anti-malarial drugs. We will combine the power of genetic manipulation and metabolomic analysis in this investigation to assess contributions made by the two branches of the unusual TCA metabolism of the parasite. We will also examine the role mitochondrial processes during in vivo infection as well as in sexual and mosquito stages of malaria parasites. Finally, we will investigate the unusual ATP synthase as well as mitochondrial respiratory complexes to understand their functional significance and impact on parasite physiology. These studies have the potential to be highly valuable in developing strategies for chemotherapeutic intervention affecting validated targets of malaria parasites. PUBLIC HEALTH RELEVANCE: There is a dire need to develop new anti-malarial drugs that target novel physiological processes of malaria parasites. The unusual mitochondrion of the malaria parasite represents an attractive target that has been validated for anti-malarial drug action. This project aims to derive deeper understanding of additional unusual features of the parasite mitochondrion with a view to eventually guide further strategies for drug development.